The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens.

Drug addiction therapies commonly fail because continued drug use promotes the release of excessive and pleasurable dopamine levels. Because the connection between pleasure and drug use becomes hard-wired in the nucleus accumbens (NAc), which interfaces motivation, effective therapies need to modulate this mesolimbic reward system. Here, we report that mice with knockdown of the cation channel TRPA1 (transient receptor potential ankyrin 1) were resistant to the drug-seeking behavior and reward effects of cocaine compared to their wildtype litter mates. In our study, we demonstrate that TRPA1 inhibition in the NAc reduces cocaine activity and dopamine release, and conversely, that TRPA1 is critical for cocaine-induced synaptic strength in dopamine receptor 1-expressing medium spiny neurons. Taken together, our data support that cocaine-induced reward-related behavior and synaptic release of dopamine in the NAc are controlled by TRPA1 and suggest that TRPA1 has therapeutic potential as a target for drug misuse therapies.

Sequestration of Gßγ by deubiquitinated arrestins into the nucleus as a novel desensitization mechanism of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to a rapid attenuation of responsiveness that occurs with repeated or continuous exposure to agonists. GRK-mediated phosphorylation and subsequent binding with arrestins in the activated receptor cytoplasmic cavity in competition with G proteins has been suggested as the conventional mechanism of desensitization. Along with widely accepted conventional mechanism of desensitization, studies of various GPCRs including dopamine D2-like receptors (D2R, D3R, D4R) have suggested the existence of another desensitization mechanism. In this study, loss-of-function approaches and D2-like receptor mutants that display different desensitization properties were used to elucidate the molecular mechanisms responsible for desensitization. RESULTS: Desensitization development entailed the signaling cascade composed of Src, PDK1, and Akt, the latter of which in turn interacted with USP33, an arrestin deubiquitinase, to promote arrestin deubiquitination. The deubiquitinated arrestin subsequently formed a complex with Gßγ and translocated to the nucleus via an importin complex, wherein it sequestered Gßγ from the receptor and Gα, thereby attenuating receptor signaling. As in D2-like receptors, both USP33 and importin ß1 were involved in the desensitization of the ß2 adrenoceptor. CONCLUSIONS: In addition to the conventional mechanism of desensitization, which occurs on the plasma membrane and in the cytosol, this study provides a new insight that another desensitization pathway in which nuclear trafficking plays a critical role is operating. It is plausible that multiple, complementary desensitization measures are in place to properly induce desensitization depending on receptor characteristics or the surrounding environment. Video Abstract.

Unveiling the Differences in Signaling and Regulatory Mechanisms between Dopamine D2 and D3 Receptors and Their Impact on Behavioral Sensitization.

Dopamine receptors are classified into five subtypes, with D2R and D3R playing a crucial role in regulating mood, motivation, reward, and movement. Whereas D2R are distributed widely across the brain, including regions responsible for motor functions, D3R are primarily found in specific areas related to cognitive and emotional functions, such as the nucleus accumbens, limbic system, and prefrontal cortex. Despite their high sequence homology and similar signaling pathways, D2R and D3R have distinct regulatory properties involving desensitization, endocytosis, posttranslational modification, and interactions with other cellular components. In vivo, D3R is closely associated with behavioral sensitization, which leads to increased dopaminergic responses. Behavioral sensitization is believed to result from D3R desensitization, which removes the inhibitory effect of D3R on related behaviors. Whereas D2R maintains continuous signal transduction through agonist-induced receptor phosphorylation, arrestin recruitment, and endocytosis, which recycle and resensitize desensitized receptors, D3R rarely undergoes agonist-induced endocytosis and instead is desensitized after repeated agonist exposure. In addition, D3R undergoes more extensive posttranslational modifications, such as glycosylation and palmitoylation, which are needed for its desensitization. Overall, a series of biochemical settings more closely related to D3R could be linked to D3R-mediated behavioral sensitization.

The tyrosine phosphorylation of GRK2 is responsible for activated D2R-mediated insulin resistance.

Dopamine D2 receptor (D2R) plays a key role in the regulation of glucose homeostasis by stimulating the secretion of many glucoregulatory hormones. Insulin resistance (IR) is associated with the pathogenesis of metabolic disorders which occurs when PI3K/Akt signaling pathway is downregulated. However, the potential involvement of D2R in insulin resistance remains unclear. In the present study, we investigated the regulation of glucose transport by D2-like receptors and discovered that activation of D2R, but not D3R or D4R, suppressed insulin-induced 2-DOG uptake and Glut4 membrane translocation in a GRK2- and Src-dependent manner. Further study revealed that activation of D2R inhibits insulin-induced phosphorylation of Akt at Thr308 and Ser473, which are hallmarks of its kinase activity, by increasing the interaction of tyrosine phosphorylated GRK2 with Akt and then preventing Akt from interacting with PDK1. Thus, this study demonstrates that Src mediated GRK2 tyrosine phosphorylation is an essential physiological event that mediates the roles of D2R in insulin resistance.

New designer phenethylamines 2C-C and 2C-P have abuse potential and induce neurotoxicity in rodents.

2C (2C-x) is the general name for the family of phenethylamines containing two methoxy groups at the 2 and 5 positions of the benzene ring. The abuse of 2C family drugs has grown rapidly, although the abuse potential and neurotoxic properties of 2C drugs have not yet been fully investigated. In this study, we investigated the abuse potential and neurotoxicity of 4-chloro-2,5-dimethoxyphenethylamine (2C-C) and 2,5-dimethoxy-4-propylphenethylamine (2C-P). We found that 2C-C and 2C-P produced conditioned place preference in a dose-dependent manner in mice, and increased self-administration in rats, suggesting that 2C-C and 2C-P have abuse potential. To investigate the neurotoxicity of 2C-C and 2C-P, we examined motor performance and memory impairment after high doses of 2C-C and 2C-P. High doses of 2C-C and 2C-P decreased locomotor activity, rota-rod performance, and lower Y-maze test, novel objective recognition test, and passive avoidance test scores. We also observed that 2C-C and 2C-P affected expression levels of the D1 dopamine receptor, D2 dopamine receptor, dopamine transporter, and phospho-dopamine transporter in the nucleus accumbens and the medial prefrontal cortex, and increased c-Fos immuno-positive cells in the nucleus accumbens. Moreover, high doses of 2C-C and 2C-P induced microglial activation, which is involved in the inflammatory reaction in the striatum. These results suggest that 2C-C and 2C-P have abuse potential by affecting dopaminergic signaling and induce neurotoxicity via initiating neuroinflammation at high doses.

Roles of the Functional Interaction between Brain Cholinergic and Dopaminergic Systems in the Pathogenesis and Treatment of Schizophrenia and Parkinson's Disease.

Most physiologic processes in the brain and related diseases involve more than one neurotransmitter system. Thus, elucidation of the interaction between different neurotransmitter systems could allow for better therapeutic approaches to the treatments of related diseases. Dopaminergic (DAergic) and cholinergic neurotransmitter system regulate various brain functions that include cognition, movement, emotion, etc. This review focuses on the interaction between the brain DAergic and cholinergic systems with respect to the pathogenesis and treatment of schizophrenia and Parkinson's disease (PD). We first discussed the selection of motor plans at the level of basal ganglia, the major DAergic and cholinergic pathways in the brain, and the receptor subtypes involved in the interaction between the two signaling systems. Next, the roles of each signaling system were discussed in the context of the negative symptoms of schizophrenia, with a focus on the α7 nicotinic cholinergic receptor and the dopamine D1 receptor in the prefrontal cortex. In addition, the roles of the nicotinic and dopamine receptors were discussed in the context of regulation of striatal cholinergic interneurons, which play crucial roles in the degeneration of nigrostriatal DAergic neurons and the development of L-DOPA-induced dyskinesia in PD patients. Finally, we discussed the general mechanisms of nicotine-induced protection of DAergic neurons.

GRK2-mediated receptor phosphorylation and Mdm2-mediated ß-arrestin2 ubiquitination drive clathrin-mediated endocytosis of G protein-coupled receptors.

Clathrin-mediated and caveolar endocytic pathways represent the major routes through which G protein-coupled receptors (GPCRs) could be internalized. GPCR kinase 2 (GRK2) and ß-arrestins are representative proteins that mediate the GPCR endocytosis. However, the molecular mechanisms through which GRK2 and ß-arrestin mediate clathrin-mediated and caveolar endocytosis remain unclear. In this study, we determined the cellular components and processes that mediate the selective interaction between clathrin/caveolin1 and GRK2/ß-arrestins. For this we utilized the following: (i) mutant dopamine D2 receptor and ß2 adrenoceptor in which the potential GRK2 phosphorylation sites were altered and (ii) cells in which clathrin, caveolin1, ß-arrestins, or Mdm2 expression were knocked down. Our results showed that clathrin-mediated endocytosis occurs more rapidly than caveolar endocytosis. Clathrin-mediated endocytosis and the interaction between clathrin and GRK2/ß-arrestin2 occurred in a GRK2-mediated receptor phosphorylation-dependent manner. In contrast, caveolar endocytosis and the interaction between caveolin1 and GRK2/ß-arrestin2 were independent of receptor phosphorylation status. Mdm2-mediated ubiquitination of ß-arrestin, which occurred in a receptor phosphorylation-dependent manner, was required for the interaction of arrestin with clathrin. Thus, this study shows that GRK2-mediated receptor phosphorylation accompanied by ß-arrestin ubiquitination is a critical cellular event that links GRK2 and ß-arrestins to clathrin-mediated endocytosis.

Cytoplasmic recruitment of Mdm2 as a common characteristic of G protein-coupled receptors that undergo desensitization.

Desensitization of G protein-coupled receptors (GPCRs) represents a gradual attenuation of receptor responsiveness by continuous or repeated exposure to agonists. The most widely accepted molecular mechanism responsible for desensitization is that of GRK2-mediated receptor phosphorylation followed by association with ß-arrestins. However, in most cases, this mechanism cannot explain the desensitization of GPCRs. In this study, we investigated whether there exists a direct correlation between desensitization and certain cellular events that commonly observed with desensitizing receptors. Our study showed that constitutive ubiquitination of ß-arrestin, accompanied by nuclear to cytoplasmic translocation of Mdm2, was observed in cells expressing desensitizing GPCRs (dopamine D3 receptor, K149C-dopamine D2 receptor, ß2 adrenoceptor, and lysophosphatidic acid receptor 1). In contrast, Mdm2 was observed in the nucleus in cells expressing non-desensitizing GPCRs (dopamine D2 receptor, C147K-dopamine D3 receptor, and dopamine D4 receptor). Molecular manipulation to convert the characteristics of the dopamine D4 receptor from non-desensitizing to desensitizing changed the status of subcellular localization of Mdm2 from nuclear to cytoplasmic. With repeated agonist treatments of desensitizing receptors, Mdm2 translocated from cytoplasm to nucleus, resulting in the deubiquitination of ß-arrestins. This study suggests that the property of a receptor that causes a change in subcellular localization of Mdm2, from the nuclear to cytoplasmic, could be used as a biomarker to predict the desensitization of a receptor.

A novel molecular mechanism responsible for phosphorylation-independent desensitization of G protein-coupled receptors exemplified by the dopamine D3 receptor.

GRK-mediated receptor phosphorylation followed by association with ß-arrestins has been proposed to be the molecular mechanism involved in the desensitization of G protein-coupled receptors (GPCRs). However, this mechanism does not explain the desensitization of some GPCRs, such as dopamine D3 receptor (D3R), which does not undergo GRK-mediated phosphorylation. Loss-of-function approaches and mutants of dopamine D2 receptor and D3R, which exhibit different desensitization properties, were used to identify the cellular components and processes responsible for desensitization. D3R mediated the recruitment of Mdm2 to the cytosol, which resulted in the constitutive ubiquitination of ß-arrestin2 in the resting state. Under desensitization conditions, cytosolic Mdm2 returned to the nucleus, resulting in the deubiquitination of cytosolic ß-arrestins. Deubiquitinated ß-arrestins formed a tight complex with Gßγ, thereby sequestering it, causing interference in D3R signaling. In conclusion, this study shows that ß-arrestins, depending on their ubiquitination status, control the G protein cycling by regulating their interactions with Gßγ. This is a novel mechanism proposed to explain how certain GPCRs can undergo desensitization without receptor phosphorylation.

ß-Arrestin2 directly or through GRK2 inhibits PKCßII activation in a ubiquitination-dependent manner.

The GRK/ß-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and ß-arrestins on PKC activation. The roles of GRK2 and ß-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D2R, D3R, and ß2 adrenergic receptor (ß2AR). The subdomains involved in the mutual interactions among GRK2, ß-arrestin2, and PKCßII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, ß-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and ß-arrestin2 mutually inhibited the PKCßII autophosphorylation, a hallmark of PKCßII activation. ß-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as ß-arrestin2. Furthermore, GRK2 facilitated ß-arrestin2 ubiquitination, thus to enhance the inhibitory actions of ß-arrestin2 on PKCßII activity. Aforementioned processes were also involved in the GRK2/ß-arrestin2-mediated inhibition of the D2R, D3R, and ß2AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of ß-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs.

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling.

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D3 receptor (D3R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D3R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gßγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gßγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gßγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D3R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D3R, which can be extended to the crosstalk between other RTKs and GPCRs.

α4ß2 nicotinic acetylcholine receptor downregulates D3 dopamine receptor expression through protein kinase C activation.

Receptor transactivation or crosstalk refers to instances in which the signaling of a given receptor is regulated by different classes of receptors. Functional crosstalk between α4ß2 nicotinic acetylcholine receptor (nAChR) and D3 dopamine receptor (D3R) that belong to the family of ligand-gated ion channels and G protein-coupled receptors, respectively, has been reported from brain dopaminergic neurons. For example, D3R is involved in the development of reward-related behaviors induced by α4ß2 nAChR stimulation. However, the molecular mechanisms involved in their crosstalk remain unclear. Among PKC isoforms (α, ßII, γ, and δ) evaluated in this study, PKCßII interacted with D3R and potentiated D3R endocytosis. Following α4ß2 nAChR stimulation, activated PKCßII translocated to the plasma membrane to induce clathrin-mediated endocytosis of D3R, resulting in downregulation and signal inhibition. Considering that D3R plays important roles in mediating reward-related physiological actions of α4ß2 nAChR, this study could provide a new insight into the regulatory mechanism involved in nicotine addiction.

Design, synthesis and docking study of 4-arylpiperazine carboxamides as monoamine neurotransmitters reuptake inhibitors.

Rational drug design method has been used to generate 4-arylpiperazine carboxamides in an effort to develop safer, more potent and effective monoamine neurotransmitters reuptake inhibitors. Out of twenty-seven synthesized compounds, compound 9 displayed potent monoamine neurotransmitter reuptake inhibitory activity against HEK cells transfected with hSERT or hNET. A Surflex-Dock docking model of 9 was also studied.

Design, synthesis, and systematic evaluation of 4-arylpiperazine- and 4-benzylpiperidine napthyl ethers as inhibitors of monoamine neurotransmitters reuptake.

Two series of 4-arylpiperazine- and 4-benzylpiperidine naphthyl ethers were designed based on structure-activity relationship (SAR) and docking model of reported monoamine neurotransmitters reuptake inhibitors. The compounds were synthesized in 3-simple steps and their biological activities were evaluated. Several compounds were proven to be potent inhibitors of serotonin and norepinephrine reuptake. Computer docking was performed to study the interaction of the most potent compound 35 with human serotonin transporter. The results of the analyses suggest that 4-arylpiperazine- and 4-benzylpiperidine naphthyl ethers might be promising antidepressants worthy of further studies.

RalA employs GRK2 and ß-arrestins for the filamin A-mediated regulation of trafficking and signaling of dopamine D2 and D3 receptor.

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D2 and D3 receptors (D2R, D3R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D2R and D3R, comparative studies were conducted on the signaling and intracellular trafficking of the D2R and D3R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D2R and D3R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D2R and D3R, through GRK2 and ß-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D2R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D2R. Knockdown of FLNA or coexpression of active RalA prevented D3R from coupling with G protein. The selective involvement of GRK2- and ß-arrestins in the RalA-mediated cellular processes of the D2R and D3R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D2R and D3R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and ß-arrestins.

Palmitoylation on the carboxyl terminus tail is required for the selective regulation of dopamine D2 versus D3 receptors.

Dopamine D2 receptor (D2R) and D3 receptor (D3R) possess highly conserved amino acid sequences but this study showed that D3R was more extensively palmitoylated than D2R. Based on this finding, the molecular basis of this selective palmitoylation of D3R was determined and the roles of palmitoylation in the regulation of D3R functions were investigated. D3R was palmitoylated on the cysteine residue on its carboxyl terminus tail, the last amino acid residue of D3R, and an exchange of the carboxyl terminus tail between D2R and D3R (D2R-D3C and D3R-D2C) resulted in the switching of the palmitoylation phenotype. When the consensus site for palmitoylation was mutated or the palmitoylation of D3R was inhibited by treatment with 2-bromopalmitate (2BP), a palmitoylation blocker, cell-surface expression, PKC-mediated endocytosis, agonist affinity, and agonist-induced tolerance of D3R were all inhibited. However, these changes were not observed when D3R palmitoylation was inhibited by replacing its carboxyl tail with that of D2R (D3R-D2C) or when the palmitoylation of D2R-D3C was inhibited by treatment with 2BP. Overall, this study shows that D3R is palmitoylated more extensively than D2R even though the carboxyl terminus tails of D2R and D3R are highly homologous, and thus provides a new clue regarding the consensus sequence for palmitoylation. This study also shows that palmitoylation controls various functionalities of D3R only when the receptor is in the intact D3R configuration.

Agonist-induced changes in RalA activities allows the prediction of the endocytosis of G protein-coupled receptors.

GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gßγ. These results suggest that agonist-induced Gßγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs.

The EGF receptor inhibits the signaling of dopamine D3 receptor through the phosphorylation of GRK2 on tyrosine residues.

Receptor transactivation or crosstalk are terms referring to instances in which the signaling of a given receptor is regulated by a different class of receptor. The epidermal growth factor (EGF) and the dopaminergic systems in the brain are closely related to schizophrenia with respect to both etiology and treatment. Thus, we investigated the functional interactions between the EGF receptor (EGFR), which belongs to the receptor tyrosine kinase family, and the dopamine D2-like receptors (D2R, D3R, and D4R), which are members of the G protein-coupled receptor (GPCR) family. Among D2-like receptors, the signaling of D3R was selectively inhibited by EGFR stimulation. Moreover loss-of-function assays showed that tyrosine-phosphorylated GRK2 mediates this inhibition by acting on the second intracellular loop of D3R. Considering that both EGFR and D3R are closely related to schizophrenia, this study could provide new molecular insight into the etiology of the disorder.

Design, synthesis and in vitro activity of 1,4-disubstituted piperazines and piperidines as triple reuptake inhibitors.

Monoamine transporters regulate the concentration of monoamine neurotransmitters, which are essential for vital physiological processes, and their dysfunction can cause several central nervous system diseases. Monoamine transporters currently appear to be the potential target in the management of these disorders. In this study, homologation and bioisosterism techniques have been used in the designing of new 1,4-disubstituted piperazines and piperidines. These derivatives were synthesized and evaluated as potential triple reuptake inhibitors for studying the structure-activity relationships. The most advanced compound, 1-(4-(5-benzhydryl-1H-tetrazol-1-yl)butyl)-4-(3-phenylpropyl)piperazine (2i), was able to inhibit monoamine neurotransmitter reuptake in an in vitro test (IC50=158.7nM for 5-HT, 99nM for NE and 97.5nM for DA). These novel potent triple reuptake inhibitor-based 1,4-disubstituted piperazine and piperidine scaffolds deserve further systematic optimization and pharmacological evaluation.

Triple reuptake inhibitors: Design, synthesis and structure-activity relationship of benzylpiperidine-tetrazoles.

Monoamine transporters are important targets in the treatment of various central nervous disorders. Several limitations of traditional reuptake inhibitors, like delayed onset of action, insomnia, and sexual dysfunction, have compelled the search for safer, more effective compounds. In this study, we have sought to identify novel monoamine reuptake inhibitors. Based upon the docking study of compounds that we had reported previously, aromatic rings (A1) were modified to generate a novel series of benzylpiperidine-tetrazoles. Thirty-one compounds were synthesized and evaluated for their triple reuptake inhibition of serotonin, norepinephrine and dopamine. Triple reuptake inhibitor, compound 2q, in particular, showed potent serotonin reuptake inhibition, validating our design approach.